The initiation of sporulation and the production of antibiotics in gram- positive sporulating soil bacteria such as Bacillus subtilis occurs in response to nutrient limitation. However, nutrient limitation also activates the synthesis of a number of degradative pathways which provide nutrients for growth and adaptation during environmental stress. To begin to understand the network of metabolic signals and systems that regulate gene expression during nutrient-limited growth in this bacterium, regulation of the B. subtilis histidine utilization enzymes (hut) is being studied. Expression of the hut enzymes is controlled by three independent regulatory systems, histidine induction, carbon catabolite repression and amino acid repression. In contrast to the Klebsiella areogenes hut enzymes, hut expression in B. subtilis is not significantly derepressed by nitrogen-limited growth. The location of the cis-acting hut sites required for regulation by these three systems was identified by deletion analysis. Localization mutagenesis will be used to more precisely define these sites. The possibility that DNA looping is involved in catabolite repression of hut expression will be examined with (i) in vivo footprinting experiments, and (ii) by determining whether mutations that alter the spacing between the two cis-acting catabolite repression sites also affects the level of catabolite repression. transacting factors mediating catabolite repression and amino acid repression will be identified by isolating mutants with altered hut expression during growth on medium containing either repressing carbon sources or amino acids. DNA complementing these mutants will be cloned and sequenced. These studies should help define the paradigms that regulate gene expression in soil bacteria. In addition, they will provide both information and genetic tools for optimizing production of medically and agriculturally important compounds in these bacteria by commercial fermentation or during growth in the soil, where nutrients are relatively scarce.